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Assessing glucose uptake through the yeast hexose transporter 1 (Hxt1)
Adhiraj Roy1, Angela D Dement2, Kyu Hong Cho3
1Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Science, 2300 Eye Street, NW, Washington, D. C., 20037, United States of America.
Plos One
|March 28, 2015
Summary
This study validates 2-NBDG as a fluorescent probe for measuring glucose uptake in yeast via the Hxt1 transporter. Key residues are critical for Hxt1 stability and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Glucose transport across cell membranes is vital for cellular metabolism.
- The hexose transporter family (Hxt) in yeast mediates glucose uptake.
- Understanding specific transporter functions requires precise experimental tools.
Purpose of the Study:
- To investigate glucose uptake using the fluorescent analog 2-NBDG in Saccharomyces cerevisiae.
- To characterize the role of yeast hexose transporter 1 (Hxt1) in glucose transport and cellular response.
- To identify critical residues within Hxt1 essential for its stability and function.
Main Methods:
- Utilized 2-NBDG, a non-metabolizable fluorescent glucose analog, to measure uptake.
- Employed a yeast hxt null strain and complemented it with wild-type and mutant Hxt1.
- Assessed glucose-dependent growth complementation and protein degradation.
- Analyzed mutant Hxt1 proteins (Hxt1N370A, HXT1W473A) for stability and function.
Main Results:
- 2-NBDG uptake was observed only in the presence of functional Hxt1, confirming its role in transport.
- Mutations at putative glucose-binding sites abolished 2-NBDG uptake and growth complementation.
- Hxt1 undergoes glucose starvation-induced degradation, with specific mutants (Hxt1N370A, HXT1W473A) showing resistance.
- Hxt1N370A mutant exhibited impaired 2-NBDG uptake and failed to rescue growth defects.
Conclusions:
- 2-NBDG is a reliable fluorescent probe for assessing glucose transport in yeast.
- Residue N370 in Hxt1 is crucial for both transporter stability and glucose uptake function.
- Specific mutations can confer resistance to Hxt1 degradation, impacting its cellular localization and activity.
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